Cell-reporter systems can exhibit cross-reactivity and microbial interference with non-target organisms. For example, if an Enterobacteriaceae reporter is used to detect E. coli in a stool sample; other species of Enterobacteriaceae such as K. pneumoniae may produce a cross-reactive signal resulting in a false positive result. Furthermore, species of other Family of bacteria, such as P. aeruginosa, A. baumannii, and S. maltophilia, which may be present in a sample, may result in microbial interference resulting in a false negative result.
Antimicrobial susceptibility tests (AST) measure the response of a microorganism to an antimicrobial and are used to determine if the microorganism is susceptible or non-susceptible to the antimicrobial. The response of a microorganism to an antimicrobial may be due to a variety of mechanisms, all of which give the same response or phenotype. For example, in carbapenem resistant Enterobacteriaceae (CRE), resistance to carbapenem antibiotics may be due to a variety of carbapenemases encoded by different genes and gene variants including blaNDM-1, blaKPC, blaIMP, blaVIM, blaCMY, etc. as well as situations that result in a carbapenem non-susceptible phenotype despite the lack of a carbapenemase such as non-carbapenemase β-lactamase hyper-expression and mutations that result in decreased uptake of a carbapenem into a cell (e.g. porin mutations).
AST is not capable of discriminating between different resistance mechanisms that impart a common phenotypic response. When testing the response of an Enterobacteriaceae to meropenem, for example, if it is found that the Enterobacteriaceae is resistant to meropenem, it cannot be determined from this assay if the resistance is due to blaNDM-1 or blaKPC, or other carbapenem resistance mechanisms.
Extensions of AST have been developed to provide limited information about the mechanism that imparts a resistance phenotype in a microorganism. For example, when conducting AST testing on Enterobacteriaceae using Amoxicillin, if the organism is found to be resistant to Amoxicillin but susceptible to Amoxicillin in the presence of clavulanic acid, a β-lactamase inhibitor, this result can indicate that a β-lactamase is linked to the Amoxicillin-resistance phenotype. However, this technique only informs of the role of a β-lactamase but not the identity of the specific β-lactamase.
Nucleic acid amplification techniques such as polymerase chain reaction (PCR) can be employed to determine the presence of specific genes that may impart a resistance phenotype in an organism. However, these techniques cannot distinguish between viable and non-viable organisms leading to possible false-positive results and also cannot determine if the detected gene is expressed and are thus incapable of measuring the phenotypic response of an organism to an antimicrobial and thus cannot generally determine antimicrobial susceptibility.
Due to these limitations, there is a need for a means of determining the underlying mechanism that imparts a phenotypic response in an organism. The identity of the specific underlying mechanism that imparts a given phenotype can be important information for epidemiological analysis and other related analyses.